X-ray Microanalysis of Ion Distribution in Abutilon Nectary Hairs

Robards, A. W. and Oates, K. 1986. X-ray microanalysis of iondistribution in Abutilon nectaryhairs—J. exp. Bot. 37:940–946. X-ray microanalysis of frozen-hydrated Abutilon nectary hairsshows that potassium is present at easily detectable levelsin all cells between the phloem and the trichome tip cells.There is little overall variation in the amount of potassiumfrom cell to cell although it was found that: (i) some basalcells accumulate potassium to a significantly higher level thanthe hair cells; and (ii) there is more potassium in the cellstowards the distal end of the trichome. These findings wouldbe consistent with a model for secretion envisaging a trans-membraneloading of nectar into a ‘secretory reticulum’ inall trichome cells. This loading process would be selectiveand would exclude ions. A pronounced increase in the level ofchlorine from the basal cell along the hair was observed. Nocomplementary cation was detected but equivalent changes inthe level of Na would have been below the detection limit ofthe system. Key words: Abutilon, nectary hairs, X-ray microanalysis, ion distribution     

Preparation techniques for electron probe microanalysis of cells

Abstract

Electron probe X-ray microanalysis can be used to determine the elemental composition of cells and tissues in defined functional states. However, reliable quantitative data can be expected only after appropriate cryopreparation techniques. The most versatile preparation and analysis technique can be outlined by four succeeding steps: 1. Cryofixation, 2. Cryoultramicrotomy, 3. Cryotransfer including freeze-drying, 4. Energy dispersive X-ray microanalysis.     

Electron-microscopic and microprobe analyses on the pigmented and unpigmented enamel of Sorex (Insectivora)

Summary Sorex belongs to the Insectivora and has a pigmented tooth enamel due to iron. The pigmented enamel (PE) has a mean Ca/P weight ratio, analyzed by quantitative electronprobe X-ray microanalysis, of about 1.9 (mean molar Ca/P ratio 1.46), and the unpigmented enamel (UE) a Ca/P weight ratio of about 2.0 (mean molar Ca/P ratio 1.59). The PE has a higher iron content (with a value of about 8%) than the UE, as shown by microanalysis of ultrathin sections. Laser microprobe mass analysis (LAMMA) has shown that the carbonate content in the UE is higher than in the PE. In the LAMMA spectrum of the negatively charged ions the carbonate lines could be compared directly with those of negatively charged iron ions. The pigmentation is associated with a low Ca/P ratio but may transfer mechanical strength and acid resistance strength to the PE.     

Functional dissimilarity of melanomacrophage centres in the liver and spleen from females of the teleost fish <Emphasis Type="Italic">Prochilodus argenteus</Emphasis>

Melanomacrophage centres (MMCs) are formed by macrophage aggregates containing pigments such as hemosiderin, melanin and lipofuscin. MMCs are found in animals such as reptiles, amphibians and, mainly, fishes, in organs such as the kidney, spleen, thymus and liver. In teleost fish, several functions have been attributed to MMCs, including the capture and storage of cations, the phagocytosis of cellular debris and immunological reactions. As the use of MMCs has been suggested as a tool for the assessment of environmental impacts, our aim has been to describe the various metabolic processes performed by MMCs in diverse organs (liver and spleen) by using the teleost Prochilodus argenteus as an animal model. MMCs from the liver and spleen were assessed by histochemistry, transmission electron microscopy, scanning electron microscopy, X-ray microanalysis techniques and biochemical assay for N-acetylglucosaminidase activity. The data showed metabolic differences in MMCs between the liver and spleen of P. argenteus in their morphometric characteristics and biochemical and elemental composition. The implications of these findings are discussed, focusing on their role in organ metabolism.     

X-ray microanalysis of freeze-dried digestive mucus in Anguilla anguilla L: modifications of ion content during the early steps of secretion

Summary— Digestive mucus of sea-water adapted eels has been observed and analyzed by the scanning electron microscope (SEM) after rapid freezing at liquid nitrogen temperature followed by freeze-drying. No chemical procedures were used in this technique. This allowed the maintenance of the mucous coating. Preliminary X-ray microanalysis carried out on freeze-fractured and freeze-dried samples of the oesophagus showed a decrease of K⁺ and an increase of Ca²⁺ and Cl^? from the basal part of the mucous cell towards its the apical part. This technique has proven to be satisfactory for it prevents translocation and loss of diffusible elements in situ and allows X-ray microanalysis in the SEM.     

The role of secondary ion mass spectrometry (SIMS) in biological microanalysis: technique comparisons and prospects.

The virtues and limitations of SIMS ion microscopy are compared with other spectroscopic techniques applicable to biological microanalysis, with a special emphasis on techniques for elemental localization in biological tissue (electron, X-ray, laser, nuclear, ion microprobes). Principal advantages of SIMS include high detection sensitivity, high depth resolution, isotope specificity, and possibilities for three-dimensional imaging. Current limitations, especially in comparison to X-ray microanalysis, center on lateral spatial resolution and quantification. Recent SIMS instrumentation advances involving field emission liquid metal ion sources and laser post-ionization will help to minimize these limitations in the future. The molecular surface analysis capabilities of static SIMS, especially with the new developments in commercial time-of-flight spectrometers, are promising for application to biomimetic, biomaterials, and biological tissue or cell surfaces. However, the direct microchemical imaging of biomolecules in tissue samples using SIMS will be hindered by limited concentrations, small analytical volumes, and the inefficiencies of converting surface molecules to structurally significant gas phase ions. Indirect detection using elemental or isotopically tagged molecules, however, shows considerable promise for molecular imaging studies using SIMS ion microscopy.     

Ionic gradients within mantle-collar epithelial cells of the land snail Otala lactea

Summary Aestivating Otala lactea have been shown to reduce the rate of evaporative water loss from the cells of the mantle-collar epithelium to a level comparable to that of an insect. X-ray microanalyses of ultrathin frozen sections from aestivating and non-aestivating snails have shown gradients of chloride and potassium ions in the apical microvillus region of the regulating mantle collar epithelium. The greatest difference in osmotic concentration occurs in the apical 2 m of the cell. There appears to be a barrier at that level that prevents water being mobilised from the underlying tissues. Methods of presenting data generated by X-ray microanalysis are also discussed.     

ELECTRON-PROBE X-RAY MICROANALYSIS: TECHNIQUES AND RECENT APPLICATIONS IN BIOLOGY

(1) X-ray microanalysis is a powerful technique, allowing the quantitative measurement of many elements of physiological interest, at physiological concentrations and with a spatial resolution typically of a few micrometres in bulk specimens, and a few hundred nanometres in thin sections. (2) The basic requirements are a focussed, high-energy electron beam, X-ray spectrometers and a means of visualizing the specimen. These facilities are found in a number of different types of commercial microanalysers, which may be based on either the transmission electron microscope, or the scanning electron microscope. Scanning microanalysers offer a lower image resolution, but are considerably more versatile than instruments based on the transmission electron microscope. (3) Preparing the specimen in a form that will withstand electron bombardment under high vacuum, and yet in which the elements to be analysed are retained in their original locations, is clearly the most critical step. For analysing diffusible elements, especially water-soluble electrolytes, the only reliable method is to freeze as rapidly as possible, and analyse the tissue without any chemical treatment. (4) The best results are obtained if frozen sections are cut and analysed on a cold-stage with their water content retained as ice. Procedures have been worked out for doing this, and for quantitative interpretation of the results, so that original tissue concentrations of the common electrolytes, phosphorus, sulphur and other elements of interest can now be measured with confidence. The original water-content of different cell and tissue compartments can also be estimated from the mass-loss on drying. (5) A simpler alternative is to freeze-dry the frozen sections before analysis. The distribution of diffusible elements is probably not too much disturbed, spatial resolution is improved, and the visual image becomes much clearer, but quantitation is made more difficult and less reliable. Nevertheless this technique is frequently used. (6) For precipitated materials and for fluid samples, much simpler methods of preparation can be used. (7) The technique is the subject of a large and rapidly expanding literature, and is providing new information on the sub-cellular distribution of electrolytes and other elements in many different tissues from animals and plants. (8) Some of the earliest applications to the study of diffusible ions were in the analysis of micropuncture samples from kidney tubules, where it has been possible to analyse many very small samples, and for several elements at once. Some preliminary information has also been obtained on the intracellular ion concentrations in kidneys subjected to different physiological conditions. (9) A particularly successful field has been the study of transporting epithelia, including vertebrate and insect digestive and excretory tissues, where the distribution of ions along the intercellular spaces has been shown not to agree with that predicted from the ‘standing gradient’ theory of osmotic coupling. The regulatory mantle epithelium in a mollusc has also been investigated, and some new information obtained on intracellular ion distributions in the frog skin. (10) Studies on nervous tissue are still at a preliminary stage, because of the structural complexity of the tissue. In muscle, however, it has been possible to demonstrate the re-uptake of calcium by intracellular structures, following its release into the cytoplasm during contraction. (11) Information has been obtained on the distribution of ions in the nuclei and chromosomes of cells at different stages in development and division. Nuclear sodium and potassium levels are generally similar to those in the cytoplasm: the very high sodium concentrations found in nuclei isolated anhydrously are shown to be artefactual. (12) A variety of plant cells has been investigated, problems of particular interest being the regulation of salt uptake by roots and leaves, and the role of potassium ions in causing opening of the stomata by osmotic swelling of the guard-cells. (13) Some applications in pathology are briefly mentioned, including studies on the fate of accidentally or deliberately introduced minerals, and on differences in the ion content of normal and diseased muscle cells. (14) Numerous observations on the subcellular distribution of calcium are collected together. In general, measurable calcium uptake by mitochondria appears to occur only in damaged cells; in muscle the cell-membrane and the sarcoplasmic reticulum seem to be the main organelles responsible for re-uptake of calcium following a contraction. Direct involvement of calcium with a contractile system has been shown in the vorticellid protozoan Zoothamnium, and a connection between calcium and exocytosis has been demonstrated in digestive and other epithelia.     

STUDY OF CELL DEATH IN FRIEND LEUKAEMIA

Cell death of splenic Friend leukaemic cells has been studied in vivo, using ¹²⁵I-UdR and ³H-TdR pulse labelling. The evolution of the splenic specific activity has been measured by autoradiography and external counting during 40 hr after injection of the labelled precursor. These two techniques show the existence of a large reutilization of ³H-TdR (50%), which is measurable as soon as 7 hr after the injection. The DNA turnover rate is rapid, 83-8 % of the splenic cellular DNA being renewed per day. These results confirm that most of the cells produced in the Friend leukaemic spleen are rapidly lost; they also demonstrate that this cell loss is mainly due to a massive death, which occurs in proerythroblastic and erythroblastic compartments after one or two cell divisions. Friend leukaemic cells, which are characterized by a limited capacity of proliferation and a short lifespan, do not appear to be malignant.     

X-ray microanalysis of apical fluid in cystic fibrosis airway epithelial cell lines.

The ionic composition of the fluid lining the airways (airway surface liquid, ASL) in healthy subjects and patients with cystic fibrosis (CF) has been a matter of controversy. It has been attempted to resolve conflicting theories by using cell cultures, but published results show a wide variety of values for the ionic concentrations in the apical fluid in these cultures. To investigate CFTR-mediated HCO(3)(-) conductance and the role of HCO(3)(-) in regulating ASL pH we determined the pH of the fluid covering the apical surface of airway epithelial cells. A normal (16HBE14o (-)) and a CF (CFBE41o (-)) bronchial epithelial cell line were grown on membrane inserts in both a liquid-liquid interface culture system for 7 days, and in an air-liquid interface culture system for one month. The elemental composition of the fluid covering the apical surface was determined by X-ray microanalysis of frozen-hydrated specimens, or by X-ray microanalysis of Sephadex beads that had been equilibrated with the apical fluid. Analysis showed that the apical fluid had a Na(+) and Cl(-) concentration of about 80-100 mM and thus was slightly hypotonic. The ionic concentrations were somewhat higher in air-liquid interface than in liquid-liquid interface cultures. The apical fluid in CF cells had significantly higher concentrations of Na and Cl than that in control cultures. In control cultures, the concentrations of Na and Cl in the apical fluid increased if glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR) was added to the apical medium. Exposing the cells to the metabolic inhibitor NaCN also resulted in a significant increase of the Na and Cl concentrations in the apical fluid. The results agree with the notion that these cell cultures are mainly absorptive cells, and that ion absorption by the CF cells is reduced compared to that in normal cells. The pH measurements of the fluid covering the apical part of cell cultures support the notion that bicarbonate ions may be transported by CFTR, and that this can be inhibited by specific CFTR inhibitors.     

Changes in Morphometry and Elemental Composition of Robinia pseudoacacia Pulvinar Motor Cells During Leaflet Movements

Changes in shape and size of Robinia pulvinar cortical cellsin relation to leaflet movements have been investigated usingan image processing system applied to drawings of transverseand longitudinal pulvinar sections. Both the size and shapeof cell sections underwent change during movement. The dorsal-leftside region of the cortex has been characterized as the extensorregion which increases turgor during opening. Morphometric changesoccur throughout the cortical motor cells except in the threeor four inner layers. K, Cl, S, and Ca distribution in cellwalls and protoplasts of inner and outer motor cells have beenmeasured with X-ray microanalysis. The distribution patternof K and Cl shows that these ions are mainly responsible forturgor changes. K and Cl were simultaneously depleted in apoplastand protoplast, which suggests that cell walls do not possessa high enough ionic reservoir during Robinia leaflet movements.Ca was always higher in flexor cell walls than in extensor regionsof closed pulvini. This fact could be related to a lower abilityto extend of flexor cells which underwent fewer morphomerticchangrs during movement.     

Role of intestinal mucus in crystal biogenesis: an electron-microscopical,diffraction and X-ray microanalytical study

Summary In the posterior intestine of the sea-water eel, mucus plays an important role in biocrystallization of calcium ions. By means of transmission and scanning electron microscopy associated with X-ray microanalysis and X-ray diffraction it has been possible to determine the role of mucous fibers as nucleation sites. Biocrystallization occurs in 2 steps: (1) Calcification of mucus. As soon as mucus is excreted in the intestinal lumen, it is loaded with calcium, as shown by lanthanum affinity and X-ray microanalysis on freeze-dried tissues. (2) Genesis of crystals. Needleshaped crystallites build up in coalescent spherites in the intestinal lumen near the microvilli. Genesis occurs as follows: (a) crystallite mineralization by nucleation in an organic matrix composed of glycoproteinaceous mucous fibers, followed by the appearance of spherites; (b) coalescence in spherites and association of spherites in rhombohedra; (c) extrusion of organic material during the final step of crystallization.     

X-ray microanalysis and chlorotetracycline staining of calcium vesicles in the green alga Mougeotia

Calcium ions have been proposed to play a key role in the sensory transduction of phytochrome-governed chloroplast movement in the green alga Mougeotia. To test this hypothesis, the intracellular pattern of calcium distribution was studied in this alga by two independent techniques, namely, X-ray microanalysis of fixed and of unfixed frozen-hydrated cells, as well as in vivo fluorescence by chlorotetracycline. Both methods of detection reveal a significant compartmentation of calcium in vesicles close to the chloroplast edge and, less frequently, in the cortical cytoplasm. Microfilaments, presumably actin, which could function in driving chloroplast movement, have been observed running between the chloroplast edge and the cortical cytoplasm (Wagner, G., Klein, K. (1978) Photochem. Photobiol. 27, 137). The vesicular calcium concentration is stable and decays only slowly in the absence of extracellular calcium much in the same way as the ability of the chloroplast to perform movements decreases. A functional relationship between vesicular calcium compartmentation and phytochrome-governed chloroplast movement in the green alga Mougeotia seems indicated.Abbreviation CTC chlorotetracycline     

The use of X-ray microanalysis to demonstrate the uptake of the molluscicide copper sulphate by slug eggs

Summary Copper has been localized in copper sulphate treated eggs of the slug Agriolimax reticulatus (Mü). This has been accomplished using a freeze-fracture technique, the freeze-dried halves of the fractured eggs being analysed in the scanning electron microscope, using both energy dispersive and wavelength dispersive X-ray microanalysis systems. The distribution of copper obtained using these methods has been compared with that achieved using a standard histochemical technique. Both techniques revealed that the copper is initially retained in the perivitelline membranes. The application of X-ray microanalysis to such studies is discussed.This research was supported by the Agricultural Research Council (G.B.), Grant No. AG 72/13     

Quantitative X-ray microanalysis of calcium in sea urchin eggs after quick-freezing and freeze-substitution

Summary Freeze-substitution was used to study the distribution of calcium in sea urchin eggs, and the validity of the technique was assessed. We followed the fate of both total and exchangeable calcium of sea urchin eggs in two species (Paracentrotus lividus and Arbacia lixula) after the various treatments needed for freeze-substitution and embedding. We compared the calcium content either by X-ray microanalysis of Epon-embedded sections of freeze-substituted eggs (6.2±0.71 mmoles/kg of Epon-embedded tissue) or by flame spectrometry analysis of living eggs (32.3±1.30 nmoles/mg protein). After standardization of units, both values lead to similar total calcium content. We also measured the movements of ⁴⁵Ca from prelabelled eggs. Exchangeable ⁴⁵Ca as well as total calcium appeared unaffected by the preparative treatment for X-ray microanalysis. In conclusion, our preparative technique for X-ray microanalysis can be considered appropriate for our material and allows us to undertake a subcellular quantification of calcium in various organelles.     

The distribution of lead in duckweed (Lemna minor L.) root tip

While considerable information on lead distribution in the cells of terrestrial plants has been collected, little is known about lead localization in the cells of the aquatic plant. Lemna minor L. (duckweed) roots were examined using X-ray microanalysis. After 1-h treatment with lead, its concentration was the highest in small vacuoles. After 6 and 12 h, the lead content of cell walls gradually increased. The changes of lead level between vacuoles and cell walls may result from redistribution of this metal from symplast to cell walls or it may reflect increased apoplastic transport. Lead was not found in the ground cytoplasm of any variants of the experiments. This fact and presence of lead in small vesicles suggests that endocytosis may play the role in lead uptake in Lemna.     

X-ray microanalysis of ion distribution within root cortical cells of the halophyte Suaeda maritima (L.) Dum.

The ion content of compartments within cortical cells of mature roots of the halophyte Suaeda maritima grown at 200 mol·m^-3 NaCl has been studied by X-ray microanalysis of freeze-substituted thin sections. Sodium and Cl were found in the vacuoles at about four-times the concentration in the cytoplasm or cell walls, whereas K was more concentrated in the cell walls and cytoplasm than in vacuoles. The vacuolar Na concentration was 12- to 13-times higher than that of K. The Na concentration of cell walls of cortical cells was about 95 mol·m^-3 of analysed volume. The cytoplasmic K concentration within the mature cortical cells was estimated to be 55 mol·m^-3 of analysed volume.     

Composition, speciation and distribution of iron minerals in Imperata cylindrica.

A comparative study of the roots, rhizomes and leaves of an iron hyperaccumulator plant, Imperata cylindrica, isolated from the banks of an extreme acidic environment, using complementary techniques: Mösbauer spectroscopy (MS), X-ray diffraction (XRD), scanning electron microscopy (SEM) coupled to energy-dispersive X-ray microanalysis (EDAX) and transmission electron microscopy (TEM), has shown that two main biominerals, jarosite and ferrihydrate-ferritin, accumulate in the different tissues. Jarosite accumulates mainly in roots and rhizomes, while ferritin has been detected in all the structures. A model of iron management in I. cylindrica is presented.     

CELL WALL STRUCTURE AND IRON DISTRIBUTION IN THE GREEN ALGA STAURASTRUM LUETKEMUELLERI (DESMIDIACEAE)1

The cell wall of Staurastrum luetkemuelleri Donnat & Ruttner was examined with scanning electron microscope (SEM) using whole cells, in thin sections with transmission electron microscope (TEM), and in air dried whole cells and unstained thin sections with X-ray microanalysis in the scanning-transmission electron microscope (STEM). The cell wall was ornamented with spines and wartlike structures. Spines were solid structures, consisting of deposits of cell wall material between two main cell wall layers. The wart-like structures were pore organs extending through the cell wall and the mucilaginous layer outside the cell wall. The pore cylinder was surrounded by deposits of cell wall material similar to the ones in the spines. X-ray microanalysis of selected areas on whole cells from a natural population showed iron accumulation in discrete locations on the cell extensions of S. luetkemuelleri. In the unstained thin sections iron was found only in the cell wall deposits in the spines. Cells grown in laboratory cultures failed to show iron accumulation regardless of readdition of iron-EDTA (Fe-EDTA) to the culture medium.     

Application of Synchrotron Radiation X-Ray Fluorescence (μ-SRXF) and X-Ray Microanalysis (SEM/EDX) for the Quantitative and Qualitative Evaluation of Trace Element Accumulation in Woody Plants

Different analytical techniques were applied to describe the localization of lead and chromium in the tissues of walnut (Juglans regia) and maple (Acer saccharinum) plants exposed to soils that had been artificially contaminated with heavy metals. Two X-ray-based techniques, synchrotron radiation X-ray fluorescence (μ-SRXF) and X-ray microanalysis (SEM/EDX), were utilized in association with induced coupled plasma optical emission spectrometry (ICP-OES). These techniques allowed the definition of maps showing a preferential accumulation of lead in the root periderm of both plants and a limited translocation of the metal to the stems. Accordingly, estimation of the lead concentration with ICP-OES showed that roots contained, on a dry weight basis, 20 to 40 times more metal than stems. Chromium, supplied to the plants as Cr³⁺ or Cr⁶⁺, was taken up only in the latter case and in limited amounts. In general, walnut was more efficient than maple in the uptake of lead and chromium. The combination of X-ray-based techniques and ICP-OES proved to be a powerful quantitative tool in mapping, with high definition, the sites of metal storage in plant tissues.